Glioma is the most common primary malignant central nervous system tumor in adult, and is usually not curable due to its invasive nature. Establishment of serum biomarkers for glioma would be beneficial both for early diagnosis and adequate therapeutic intervention.
Adachi-Hayama et al BMC Cancer 2014, 14:452 http://www.biomedcentral.com/1471-2407/14/452 RESEARCH ARTICLE Open Access Circulating anti-filamin C autoantibody as a potential serum biomarker for low-grade gliomas Masayo Adachi-Hayama1,3†, Akihiko Adachi1†, Natsuki Shinozaki1,2,3, Tomoo Matsutani1, Takaki Hiwasa2, Masaki Takiguchi2, Naokatsu Saeki1 and Yasuo Iwadate1* Abstract Background: Glioma is the most common primary malignant central nervous system tumor in adult, and is usually not curable due to its invasive nature Establishment of serum biomarkers for glioma would be beneficial both for early diagnosis and adequate therapeutic intervention Filamins are an actin cross-linker and filamin C (FLNC), normally restricted in muscle tissues, offers many signaling molecules an essential communication fields Recently, filamins have been considered important for tumorigenesis in cancers Methods: We searched for novel glioma-associated antigens by serological identification of antigens utilizing recombinant cDNA expression cloning (SEREX), and found FLNC as a candidate protein Tissue expressions of FLNC (both in normal and tumor tissues) were examined by immunohistochemistry and quantitative RT-PCR analyses Serum anti-FLNC autoantibody level was measured by ELISA in normal volunteers and in the patients with various grade gliomas Results: FLNC was expressed in glioma tissues and its level got higher as tumor grade advanced Anti-FLNC autoantibody was also detected in the serum of glioma patients, but its levels were inversely correlated with the tissue expression Serum anti-FLNC autoantibody level was significantly higher in low-grade glioma patients than in high-grade glioma patients or in normal volunteers, which was confirmed in an independent validation set of patients’ sera The autoantibody levels in the patients with meningioma or cerebral infarction were at the same level of normal volunteers, and they were significantly lower than that of low-grade gliomas Total IgG and anti-glutatione S-transferase (GST) antibody level were not altered among the patient groups, which suggest that the autoantibody response was specific for FLNC Conclusions: The present results suggest that serum anti-FLNC autoantibody can be a potential serum biomarker for early diagnosis of low-grade gliomas while it needs a large-scale clinical study Keywords: Glioma, Filamin C, FLNC, Biomarker, Early diagnosis Background Glioma is the most common type of primary brain tumor in adults Currently, tumor grading by histological analysis of surgically-resected specimens is the most reliable predictor of glioma prognosis They are classified into four grades; low-grades including WHO Grade I (localized gliomas) and WHO Grade II (diffuse gliomas), and highgrades including WHO Grades III (anaplastic gliomas) and WHO Grade IV (glioblastoma) Despite the recent * Correspondence: iwadatey@faculty.chiba-u.jp † Equal contributors Department of Neurological Surgery, Chiba University, Graduate School of Medicine, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan Full list of author information is available at the end of the article advances in glioma diagnosis and therapy, two-year survival for the grade IV glioblastoma is less than 30% Even among patients with low-grade gliomas that usually confer a relatively good prognosis, treatment is almost never curative under the current diagnostic system [1] Identification of specific glioma antigens is long awaited for the clinical management such as early diagnosis, more objective diagnosis, monitoring treatment response, and for novel therapeutic targets for glioma [2-5] In the previous study utilizing proteomics, we found several proteins that are overexpressed in high-grade gliomas and some were potentially applicable to serum biomarkers by ability of secretion [6] Serum levels of these candidate proteins © 2014 Adachi-Hayama et al.; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Adachi-Hayama et al BMC Cancer 2014, 14:452 http://www.biomedcentral.com/1471-2407/14/452 were shown to correlate significantly with tumor grade, invasive nature of the tumor and patient survival periods [7,8] However, detection of low-grade tumors is difficult by utilizing the protein amount-based diagnostic system because their serum protein levels may not be sufficiently altered to be detectable by current proteomic technologies One approach for overcoming this difficulty and thereby enable early detection of slight changes in protein amount, protein structure or protein localization would be utilization of antigen-antibody interactions [9,10] Detection of the host immune reactions which can respond to slight changes in the precursor cells that have started to transform into neoplastic cells would be a breakthrough to enable early diagnosis of cancers including glioma To verify this concept, we utilized immunoscreening of cDNA libraries prepared from glioblastoma cells with IgG in the sera from glioma patients We found filamin C (FLNC), which is normally restricted in muscle tissues but abundantly exists in fetal central nervous system [11,12], as a candidate protein for glioma antigen Filamins are an actin cross-linker, and serve as scaffolds for many binding partners including channels, receptors, intracellular signaling molecules, and transcription factors [13,14] Because of these extensive fields of associating proteins, mutations in filamin genes result in a wide range of cell and tissue anomalies Especially they have a decisive role in cellular motility and migration [11-14] Furthermore, Filamin genes mutations are common in human breast and colon cancers [15] Many recent studies have suggested filamin A as an important factor for tumor malignancy and invasiveness in various human cancers including primary brain tumors [16-20] In addition, filamin A interacts with BRCA1/2 or other DNA repair-related proteins to affect the DNA repair process resulting in resistance to radiation and chemotherapy [21,22] In this paper, we examined tissue expressions of FLNC (both in normal and tumor tissues), and investigated the serum levels of anti-FLNC autoantibody in glioma patients Methods Sera and tissue specimens We analyzed 131 glioma patients’ sera (low-grade, 72; highgrade, 59) along with 77 sera from healthy volunteers, 19 sera from patients with meningioma, and 24 patients with cerebral infarction at chronic stage These were newlydiagnosed patients, and had no other cancer or diseases at the time of sample collection They had serum drawn at the time of initial diagnosis The patient demographics and clinical profiles are presented in Table Forty-eight glioma tissues surgically-resected from newly diagnosed glioma patients (low-grade, 22; high-grade, 26) and 10 healthy brain tissues were analyzed for the tissue expression of FLNC The normal brain tissues were obtained from the patients undergoing resection of extra-axial brain tumors or epilepsy surgery Sixty-five serum samples from glioma patients and Page of 10 Table Characteristics of the training set and validation set Training set Demographic Glioma (n=65) Control (n=38) Validation set Glioma (n=66) Control (n=39) Age Mean 46.6 47.6 49.2 51.2 Range 12-74 34-68 24-78 22-77 Male 40 21 37 21 Female 25 17 29 18 Sex WHO grade I II 28 29 III 9 IV 21 20 38 samples from healthy volunteers were used to develop a diagnostic model (training set) that was validated in an independent, blinded validation set using the serum samples from 66 glioma patients and 39 healthy volunteers The protocol of this study was approved by the Institutional Review Board of Chiba University, and written informed consent was obtained from the patients or their guardians Total RNAs of the lung, liver, spleen, testis and muscle were commercially obtained (Zyagen Laboratories, San Diego, CA) Total RNAs of lung, liver, spleen, testis and muscle were commercially obtained (Zyagen Laboratories, San Diego, CA) Serological analysis of recombinant cDNA expression libraries (SEREX) Total RNA was prepared from the U87MG glioblastoma cell line by the acid guanidium thiocyanate-phenol-chloroform method, and purified to poly(A) + RNA using the OligotexdT30 (Super) mRNA Purification Kit (Takara Biochemicals, Kyoto, Japan) cDNA was ligated into the EcoRI-XhoI site of the λZAP II phage The original library size was × 106 Escherichia coli XL1-Blue MRF’ was infected with the λZAP II phages which contained the U87MG cDNA library, and the expression of cDNA was induced by blotting on nitrocellulose membranes which had been pretreated for 30 with 10 mM IPTG (Wako Pure Chemicals, Osaka, Japan) After washing and blocking, the membranes were exposed in 1:2000-diluted sera from 18 glioma patients Then, the membranes were treated with 1:5000-diluted alkaline phosphatase-conjugated F(ab)’ fragment-specific goat antihuman IgG Positive reactions were detected by incubation in a color development solution containing 0.3 mg/mL of nitroblue tetrazolium chloride and 0.15 mg/ mL of 5-bromo-4-chloro-3-indolyl-phosphate Positive clones were re-cloned twice to obtain monoclonality and retested for the serum reactivity Adachi-Hayama et al BMC Cancer 2014, 14:452 http://www.biomedcentral.com/1471-2407/14/452 Page of 10 Sequence analysis of identified antigens Extraction of mRNA and preparation of cDNA Monoclonalized phage cDNA clones were converted to pBluescript phagemids by in vivo excisions with ExAssist helper phage (Stratagene, La Jolla, CA) Plasmid DNA was obtained from E coli SOLR strain transformed by the phagemid The cDNA inserts were sequenced by the dideoxy chain termination method using the DNA sequencing kit BigDye Terminator (Applied Biosystems, Foster City, CA) Sequences were analyzed for homology with public databases of known genes and proteins using BLAST on the National Center for Biotechnology Information’s website (http://www.ncbi.nlm.nih.gov/gene or protein) The mRNAs were extracted from the tumors and normal brain tissues using the QIAzol Lysis Reagent and RNeasy® Lipid Tissue Mini Kit (QIAGEN, Tokyo, Japan), followed by DNase treatment One μg of each mRNA was reversely transcribed using the oligo dT primer (Takara Biochemicals, Inc., Tokyo, Japan) and Super Script II (Invitrogen, CA) Purification of recombinant FLNC protein The cDNA insert of FLNC incorporated in pBlueScript was cleaved by EcoRI and XhoI, and then recombined in pGEX-4 T-3 E coli JM109 cells containing either pGEX4 T-3- FLNC or control pGEX-4 T-3 were cultured in 200 mL of Luria broth and treated with mM IPTG for 2.5 hrs The cell lysate was centrifuged and GST-FLNC in the supernatant was directly purified with glutathioneSepharose (Amersham Biosciences, Piscataway, NJ) The purified proteins were concentrated using Apollo centrifugal concentrators (Orbital Biosciences, Topsfield, MA) ELISA for anti-FLNC autoantibody Fifty μl of antigen (GST or GST-tagged recombinant FLNC) was added to each well, and incubated at 4°C overnight The plate was washed and blocked with 10% fetal calf serum in PBS (PBS-FCS) Fifty μl of sera diluted at 1:100 in 10% PBS-FCS was added to the wells and then they were incubated The bound IgG antibodies were detected by incubating with horseradish peroxidase-conjugated antihuman IgG antibody (Jackson Immuno Research Laboratories, West Grove, PA), followed by the addition of 100 μl of a peroxidase substrate (o-phenylenediamine, 0.4 mg/ml) in a citrate-phosphate buffer Absorbance at 490 nm was determined using a microplate reader (Emax; Molecular Devices, Sunnyvale, CA) Real-time RT-PCR The real-time quantitative RT-PCR with SYBR-green was performed using the Light Cycler (Roche Diagnostics, Meylan, France) The amplification was performed using 5’-GGACATGAGTGGCCGGTACAC-3’ as the forward primer and 5’-ACTGTGACGAGGCACTTGCTG-3’ as the reverse primer A series of cDNA dilutions, 1/1, 1/10, 1/100, and 1/1000, were used in each run separately Standard curves were obtained by doing serial dilutions of the same sample in each run Then, mM of each primer and mM of MgCl2 in the total volume of 20 μl were used The real-time RT-PCR cycle started with the initial denaturation at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 10s, annealing at 61°C for 10s and then elongation at 72°C for 10s As an internal quantitative control of the gene expression, the glyceraldehydes3-phosphate dehydrogenase (GAPDH) gene expression was used The ratios of filamin C and GAPDH gene Sandwich ELISA for Serum FLNC measurement ELISA 96-well plates were coated with 20 μg/ml antihuman FLNC antibody, and were filled overnight with 50 μl of patients’ sera diluted 1:100 The plates were developed with o-phenylene-diamine (Sigma-Aldrich, St Louis, MO) and were read at an absorbance of 490 nm Total IgG measurement Serum total IgG was measured in the same samples as the anti-FLNC autoantibody measurements according to the manufacturer’s instructions using a human IgG ELISA quantitative kit (Bethyl, Montgomery, TX) Figure Tissue expression of filamin C mRNA was measured by quantitative real-time RT-PCR analysis in normal brain tissues, low-grade gliomas, and high-grade gliomas, and also in lung, liver spleen, testis and muscle which were commercially obtained The filamin C mRNA expression was significantly up-regulated in high-grade gliomas compared with normal brain tissues It was moderately upregulated in low-grade gliomas and normal muscle tissues The mean values of duplicate experiments for each sample are presented Adachi-Hayama et al BMC Cancer 2014, 14:452 http://www.biomedcentral.com/1471-2407/14/452 expressions represented the normalized relative levels of filamin C expressions Immunohistochemistry IHC staining was performed on μm paraffin-embedded sections Antigenicity was recovered by the microwave method Endogenic peroxidase was inactivated with 0.3% H2O2 methanol After antigen blocking, the sections were incubated overnight with mouse monoclonal primary antibody against FLNC (Lab Vision, Fremont, CA) The sections were then incubated with mouse biotinylated secondary antibody followed by the ABC complex reaction Page of 10 Finally, the reaction was visualized using DAB and counterstained with hematoxylin To quantitate FLNC protein expression, the mean percentage of positive tumor cells was determined in at least random fields at x400 magnification in each section Statistical analysis Results of ELISA were statistically analyzed by unpaired ttest Receiver–operating characteristics (ROC) curve analysis was used to determine the optimal cutoff values for differential diagnosis of low-grade gliomas and healthy volunteers The survival rates were estimated using Kaplan- A B 100 90 80 70 60 50 40 30 20 10 Figure Immunohistochemistry (IHC) for FLNC among normal brain, low-grade glioma and high-grade glioma (A) FLNC protein expression level was higher in high-grade glioma (e, f) than in low-grade glioma (c, d) which expressed significantly higher level of FLNC than normal brain tissues (a, b) FLNC expression is only observed around the nucleus of glia cells in normal brains, whereas it spreads into the whole cytoplasm and the fibrous cellular processes of the glioma cells (magnification × 400) (B) Quantitative analysis of FLNC protein expressions in IHC shows that the mean percentage of positive tumor cells gets higher as the tumor grade advances (normal brain vs low-grade glioma; p = 0.0132, low-grade glioma vs high-grade glioma; p = 0.0005) Adachi-Hayama et al BMC Cancer 2014, 14:452 http://www.biomedcentral.com/1471-2407/14/452 A Page of 10 B Figure ELISA of anti-FLNC autoantibody levels in the sera from glioma patients and normal volunteers (A) For a training set, anti-FLNC autoantibody concentration in the sera from low-grade gliomas was significantly higher than those from high-grade gliomas (p = 0.0101) or normal volunteers (p < 0.0001) (B) The ELISA result obtained in the training set was confirmed in an independent validation set (low-grade vs high-grade: p = 0.0036, low-grade vs healthy: p = 0.0010) Meier method, and they were compared with the log-rank test The correlation between the filamin C mRNA expression levels and serum anti-FLNC autoantibody concentrations was analyzed using the non-parametric Spearman’s rank test The statistical analyses were performed using Stat-View software and SAS software (SAS Institute Inc., Cary, NC) Results Screening of the patients’ sera by serological analysis of recombinant cDNA expression libraries (SEREX) resulted in identification of filamin C (FLNC) as one of the candidate glioma antigens (Additional file 1: Table S1) The list included many signal-transduction molecules and transcription factors, which was also confirmed in a previous proteomic study We first examined whether the expression of filamin C mRNA is elevated in the glioma tissues (Figure 1) Quantitative reverse transcription–PCR (qRT-PCR) analysis of various glioma tissues and normal brain tissues confirmed that filamin C mRNA expression was significantly up-regulated in low-grade gliomas compared with normal brain tissues High-grade gliomas expressed higher level of filamin C mRNA than low-grade gliomas Other normal tissues including lung, liver, spleen, and testis contained the same levels of filamin C mRNA as normal brain tissues In contrast, muscle tissues had a higher level than the other normal tissues and the same level as low-grade glomas (Figure 1) We then analyzed protein expression levels and distributions in paraffin-embedded clinical specimens utilizing semiquantitative immunohistochemical analysis (Figure 2) FLNC protein expression level was higher in high-grade glioma than in low-grade gliomas which expressed significantly higher level of FLNC than normal brain tissues (Figure 2-a, 2-b) FLNC protein expression was only observable around the nucleus of glial cells in normal brain tissue, but it spread into the whole cytoplasm and the fibrous cellular processes in the low-grade glioma cells (Figure 2-c) We measured anti-FLNC auto-antibody concentrations by ELISA in a training set consisting of 65 glioma patients (low-grade, 35; high-grade, 30) and 38 healthy volunteers focusing on FLNC (Table 1) The results showed the serum anti-FLNC autoantibody level was significantly higher in low-grade gliomas than in high-grade gliomas or healthy volunteers (low-grade vs high-grade: p = 0.0101, low-grade vs healthy: p < 0.0001) (Figure 3-a) We then analyzed the antibody level in a prospectively-collected validation set consisting of 66 glioma patients (low-grade, 37; high-grade, 29) and 39 healthy volunteers (Table 1) The significantly increased autoantibody level was confirmed in the patients with low-grade gliomas as compared to those in high-grade tumors or healthy volunteers (low-grade vs high-grade: p = 0.0036, low-grade vs healthy: p = 0.0010) (Figure 3-b) Although the categorization of grade I and grade II gliomas into low-grade gliomas is generally used in the clinical setting, these two are different diseases biologically and clinically So, the anti-FLNC autoantibody Table Sensitivity and specificity of anti-FLNC antibody n Healthy volunteers Mean value Standard p-value for deviation healthy volunteers 77 0.242 0.115 Grade I 15 0.359 0.112 0.0021 Grade II 57 0.373 0.152